Summary of Work: Repair of double-strand breaks (DSBs) in the chromosomal DNA of humans and other eukaryotic organisms involves a complex interplay between proteins associated with repair of broken DNA ends, maintenance of chromatin structure and DNA damage-sensitive checkpoints. Mutations within genes involved in the two major pathways of DSB repair, referred to as homologous recombination and nonhomologous end-joining (NHEJ), and in cell cycling responses to DNA strand breaks have been implicated in the etiology of several human cancers and in processes leading to cell senescence. Our research efforts have focused on DSB repair in the genetically tractable yeast Saccharomyces cerevisiae. Most genes comprising the two major pathways of DSB repair are structurally and functionally conserved in yeast and human cells. We have developed an in vivo system for enyzmatically generating defined DSBs and conducted a systematic analysis of genes involved in repair using multiple assay systems. We have a) provided the first demonstration that yeast genes involved in recombination-independent NHEJ play an essential role in the repair of EcoRI endonuclease-induced DSBs in vivo; b) shown that the functions of two repair complexes (involving Ku70:Ku80 and Rad50:Mre11:Xrs2) in NHEJ are genetically separable; c) developed the first functional assay for yeast DNA Ligase IV based on quantitation of endonuclease-induced checkpoint activation in dnl4 strains; d) confirmed that the mismatch repair exonuclease encoded by EXO1 and the excision repair endonuclease Rad1/Rad10 are involved in the DSB recombinational repair pathway; e) revealed that low-level expression of PvuII endonuclease (generating DSBs with blunt termini) is lethal in haploid cells, but not in diploid cells. This latter result establishes that the structures at the ends of DSBs are critical for determining their mechanism(s) of repair.